Synthesis and Herbicidal Activity of Triketone-Quinoline Hybrids as Novel 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors

Article abstract of DOI:10.1021/acs.jafc.5b01530

4-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) is one of the most important targets for herbicide discovery. In the search for new HPPD inhibitors with novel scaffolds, triketone-quinoline hybrids were designed and subsequently optimized on the basis of the structure-activity relationship (SAR) studies. Most of the synthesized compounds displayed potent inhibition of Arabidopsis thaliana HPPD (AtHPPD), and some of them exhibited broad-spectrum and promising herbicidal activity at the rate of 150 g ai/ha by postemergence application. Most promisingly, compound III-l, 3-hydroxy-2-(2-methoxy-7-(methylthio)quinoline-3-carbonyl)cyclohex-2-enone (K_i = 0.009 ～M, AtHPPD), had broader spectrum of weed control than mesotrione. Furthermore, compound III-l was much safer to maize at the rate of 150 g ai/ha than mesotrione, demonstrating its great potential as herbicide for weed control in maize fields. Therefore, triketone-quinoline hybrids may serve as new lead structures for novel herbicide discovery.

Full text of DOI:10.1021/acs.jafc.5b01530

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Article
Syntheses and Herbicidal Activity of Triketone-Quinoline Hybrid
as Novel 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors
Da-Wei Wang, Hong-Yan Lin, Run-Jie Cao, Tao Chen, Feng-Xu Wu,
Ge-Fei Hao, Qiong Chen, Wen-Chao Yang, and Guang-Fu Yang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01530 • Publication Date (Web): 26 May 2015
Downloaded from http://pubs.acs.org on May 30, 2015
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Syntheses and Herbicidal Activity of Triketone-Quinoline Hybrid as Novel
4-Hydroxyphenylpyruvate Dioxygenase Inhibitors
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DaꢀWei Wang,† HongꢀYan Lin,† RunꢀJie Cao, † Tao Chen,† FengꢀXu Wu,† GeꢀFei Hao,† Qiong
*
‡
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Chen,† WenꢀChao Yang,† and GuangꢀFu Yang†,
†Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of
Chemistry, Central China Normal University, Wuhan 430079, P. R. China
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‡Collaborative Innovation Center of Chemical Science and Engineering, Tianjing 30071, P.R.China
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*corresponding authors:
Eꢀmail: qchen@mail.ccnu.edu.cn
Tel: +86ꢀ27ꢀ67867800, Fax: +86ꢀ27ꢀ67867141.
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ABSTRACT: 4ꢀHydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) is one of the
most important targets for herbicide discovery. To search for new HPPD inhibitors with novel
scaffolds, triketoneꢀquinoline hybrids were designed and subsequently optimized based on the
structureꢀactivity relationship (SAR) studies. Most of the synthesized compounds displayed
potent inhibition of Arabidopsis thaliana HPPD (AtHPPD), and some of them exhibited
broadꢀspectrum and promising herbicidal activity at the rate of 150 g ai/ha by postꢀemergence
application. Most promisingly, compound III-l, 3ꢀhydroxyꢀ2ꢀ(2ꢀmethoxyꢀ7ꢀ(methylthio)
ꢀquinolineꢀ3ꢀcarbonyl)cyclohexꢀ2ꢀenone (Ki = 0.009 ꢀM, AtHPPD), had broader spectrum of
weed control than mesotrione. Furthermore, compound III-l was much safer to maize at the rate
of 150 g ai/ha than mesotrione, demonstrating its great potential as herbicide for weed control in
maize field. Therefore, triketoneꢀquinoline hybrid may serve as a new lead structure for novel
herbicides discovery.
KEYWORDS: 4ꢀHydroxyphenylpyruvate dioxygenase; Herbicide; quinoline; triketone;
rational design.
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INTRODUCTION
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Modern weed management is still demanding of new herbicides that can control a wide
spectrum of weeds, including herbicideꢀresistant biotypes of these species. In addition, a new
herbicide should also be 'selective' to the crops and friendly to the environment.1, 2 Among the
known herbicide classes, 4ꢀhydroxyphenylpyruvate dioxygenase (EC 1.13.11.27,
HPPD)ꢀinhibiting herbicides can offer such solutions.3, 4 HPPD is an important enzyme in the
metabolism of tyrosine, catalyzing the conversion of 4ꢀhydroxyphenyl pyruvic acid (HPPA) into
homogentisic acid (HGA). In plants, HGA can be further transformed into tocopherols and
plastoquinone, both of them are crucial for the normal growth of plants. Inhibition of HPPD
causes a reduction in carotenoids levels, which indirectly affects photosynthesis. Then the plants
exposed to sunlight will be severely damaged, developing unique bleaching symptoms followed
by necrosis and death.5ꢀ8 HPPDꢀinhibiting herbicides have many advantages, such as
broadꢀspectrum weed control (including resistant to other herbicides), excellent crop selectivity,
benign environmental effects, low application rate and low toxicity.
Up to now, there are about 13 commercial HPPD herbicides, which are mainly classified into
three categories: triketones, pyrazoles, and isoxazoles (diketonitrile). The minimum substructure
of these inhibitors is mainly based around 2ꢀbenzoylethenꢀ1ꢀol or 2ꢀheteroaroylethenꢀ1ꢀol,
which is involved in the binding of these inhibitors to the ironII in the active site of HPPD.9, 10
Among the commercialized herbicides, triketone derivatives are one of the most widely studied,
due to the structural features of these derivatives. The structure feature of these triketones can be
further divided into two parts, triketone and aromatic moieties. Extensive studies have proved
that, modification of the aromatic part is an effective way to obtain new inhibitors with
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improved potency.3, 4, 11 Additionally, many natural and synthetic triketones have aliphatic side
chains that demonstrate the importance of lipophilicity for activity.12, 13
A series of novel triketoneꢀcontaining quinazolineꢀ2,4ꢀdiones by modifying the aromatic part
have designed previously.14ꢀ16 Most of the synthesized inhibitors showed 'good' to 'excellent'
herbicidal activity. As a follow up to discover novel HPPD inhibitors with improved potency, a
series of compounds with new heterocyclic aromatic side chains have been prepared. Quinoline
is an important structural motif of many natural products, and its derivatives are important
pharmaceutical and agrochemical intermediates.17ꢀ20 For example, in pharmaceuticals, the
derivatives of quinoline exhibited extensive biological activities, including antimalarial,
antitumor, antiꢀinfective, and antiꢀinflammatory; in agrochemicals, quinoline is a substructure of
many herbicides, fungicides and insecticides.21ꢀ23 Therefore, quinoline might be a promising
aromatic part to integrate with the triketone pharmacophore. Therefore, a series of novel
triketoneꢀquinoline hybrids was designed. Herein, we will report the detail of rational design,
synthetic chemistry, inhibitory activity against Arabidopsis thaliana HPPD (AtHPPD),
herbicidal activity, and structure–activity relationships (SAR) of triketoneꢀquinoline hybrids. As
anticipated, many of the synthesized compounds displayed nanomolar potency toward AtHPPD.
In addition, some compounds exhibited promising and broadꢀspectrum herbicidal activity at the
rate of 150 g ai/ha as well as selectivity to several crops, such as maize, wheat or rice.
MATERIALS AND METHODS
The detailed information for the synthesis of compounds IꢀVI are shown in Figures 1 to 4.
X-ray Diffraction. Compound III-d was recrystallized from a mixture of acetone and
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nꢀhexane to afford a suitable single crystal. Light brown crystals of III-d (0.20 mm × 0.20 mm ×
0.20 mm) were mounted on a quartz fiber with protection oil. Cell dimensions and intensities
were measured at 298 K on a Bruker SMART APEX DUO area detector diffractometer (Bruker
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AXS, Madison, WI) with graphite monochromated Mo Kα radiation (λ = 0.71073 Å); θmax
=
28.00; 14889 measured reflections; 4658 independent reflections (Rint = 0.0484). The data sets
were integrated and reduced using SAINT Plus Programme.24 Data were corrected for Lorentz
and polarization effects and for absorption (Tmax= 0.9821; Tmin = 0.9821). The structure was
solved by direct method using SHELXS97 and refined with SHELXL970.25 Fullꢀmatrix
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leastꢀsquares refinement based on F2 using the weight of 1/[σ2(Fo ) + (0.0976P)2 + 0.2989P]
gave final values of R1 = 0.0690,
ωR2 = 0.1652, and GOF(F) = 1.128 for 265 variables, 265
parameters and 4658 contributing reflections. Maximum shift/error
=
0.000, and
maximum/minimum residual electron density = 0.614 per ꢀ0.329 e Å−3. Hydrogen atoms were
observed and placed at their ideal positions with a fixed value of their isotropic displacement
parameter.
Compound III-f was recrystallized from a mixture of acetone and nꢀhexane to afford a
suitable single crystal. Light yellow crystals of III-f (0.12 mm × 0.10 mm × 0.10 mm) were
mounted on a quartz fiber with protection oil. Cell dimensions and intensities were measured at
298 K on a Bruker SMART APEX DUO area detector diffractometer with graphite
monochromated Mo Kα radiation (λ = 0.71073 Å); θmax = 30.00; 15593 measured reflections;
4396 independent reflections (Rint = 0.1064). The data sets were integrated and reduced using
SAINT Plus Programme.24 Data were corrected for Lorentz and polarization effects and for
absorption (Tmax= 0.9904; Tmin = 0.9885). The structure was solved by direct method using
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SHELXS97 and refined with SHELXL970.25 Fullꢀmatrix leastꢀsquares refinement based on F2
using the weight of 1/[σ2(Fo ) + (0.0886P)2 + 0.2528P] gave final values of R1 = 0.0514,
ωR2 =
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0.1438, and GOF(F) = 1.067 for 211 variables, 211 parameters and 4396 contributing reflections.
Maximum shift/error = 0.000, and maximum/minimum residual electron density = 0.869 per
ꢀ0.369 e Å−3. Hydrogen atoms were observed and placed at their ideal positions with a fixed
value of their isotropic displacement parameter.
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Crystallographic data for compounds III-d and III-f have been deposited with the Cambridge
Crystallographic Data Centre as a supplementary publication with the deposition No. 1040193
and 977357, respectively. These data can be obtained free of charge from
http://www.ccdc.cam.ac.uk/.
Plasmid Construction, Protein Expression and Purification, activity assays, and kinetic
inhibition studies. AtHPPD was constructed by PCR using cDNA of HPPD in
pMD19ꢀT Simple (Hangzhou BIOSCI Biotechnology Company, Hangzhou, China) as the
template. The primers used here were 5'ꢀCATGCCATGGGCCACCAAAACGCCGCꢀ3' (NcoI)
and 5'ꢀ CGCGGATCCTCꢀAGTGGTGGTGGTGGTGGTGTCCCACTAACTGTTTꢀ3' (BamHI).
PCR conditions were 35 cycles at 94 °C for 30 s, 55 °C for 30 s and 68 °C for 1.5 min. The
amplion was introduced into the expression vector pETꢀ15b and subsequently transformed into
E. coli BLꢀ21(DE3). The DNA sequences of the positive clones were confirmed by DNA
sequencing with Shanghai SANGON Company.
Recombinant AtHPPD was overꢀexpressed in E. coli BLꢀ21 (DE3) cells with pETꢀ15bꢀHPPD
plasmid. Recombinant homogentisate 1,2ꢀdioxygenase (HGD) from human was overꢀexpressed
in E. coli BLꢀ21 (DE3) cells with pETꢀ28aꢀHGD plasmid. The cells were grown at 37 °C in
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Luria Bertani broth supplemented with 50
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g/mL of kanamycin (pETꢀ28a plasmid) or 100
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g/mL antimycin (pETꢀ15b plasmid) according to previous publication.26 Expression of the
AtHPPD plasmid was incubated at 37 °C for 12 h. HGD was induced by 0.5 mM Isopropyl
βꢀDꢀ1ꢀthiogalactopyranoside (IPTG) when bacterial grown reached an A600 of 0.6, and then the
cells were incubated for another 40 h at 15 °C. After harvesting by centrifugation (5000 x g,
30 min), the pellet was resuspended in buffer (20 mM HEPES，20 mM NaCl，pH 7.0) and
washed twice, followed by sonication using a cell disruptor. A crude cellꢀfree supernatant was
obtained by centrifugation at 20,000 x g for 30 min.
AtHPPD was purified in two chromatographic steps. The crude cellꢀfree supernatant was
loaded onto a NiꢀNTA column (Qiagen, Canada), equilibrated with 20 mM HEPES, pH 7.0.
Then, HPPD was eluted with 20 mM HEPES, pH 7.0, 150 mM NaCl, and 250 mM imidazole.
The fractions containing HPPD were concentrated and the buffer was exchanged for 20 mM
HEPES, pH 7.0 by ultrafiltration in Ultrafree filter devices (Millipore, MA). To further purify
the recombinant HPPD, anion exchange chromatography was carried out on Q resin
(AmershamꢀPharmacia Biotech, Germany) in 20 mM HEPES, pH 7.0. Elution of the
recombinant HPPD was carried out in a linear gradient from 0 to 250 mM NaCl. Again, the
HPPDꢀcontaining fractions were collected and the buffer was exchanged to 20 mM HEPES, pH
7.0.
The in vitro activity and inhibition of AtHPPD were measured by a modification of coupled
enzyme assay methods previously reported in the literature.27 Assays were performed in
96ꢀwell plates at 30 °C using a UV/visible plate reader (Bioꢀtech, Vermont) to monitor the
formation of maleylacetoacetate at 318 nm (ε330 = 13 500/M/cm). The reaction mixture in a
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total assay volume of 200 ꢀL contained appropriate amounts of HPPA, 100 ꢀM FeSO4, 2 mM
sodium ascorbate, 20 mM HEPES buffer (pH 7.0), HPPD and HGD. Before assays were
conducted, all reaction components were preequilibrated at 30 °C for at least 10 min. The
amount of HGD activity was predetermined to be in large excess of the HPPD activity to
ensure that the reaction was tightly coupled (the Km of HGD for HGA was 25 ꢀM). Each
experiment was repeated at least three duplicates and the values were averaged. HPPD
inhibitors were dissolved in dimethyl sulfoxide (DMSO) for stock solution and diluted to
various concentrations with reaction buffer just before using. The inhibition constant (Ki), the
indication of an inhibitor’s potency, was obtained from the Dixon plot of plotting 1/v against
concentration of inhibitor at certain concentrations of substrate. Bovine serum albumin is
usually added up to 0.5% of the total reaction volume for coating of the target enzyme during
the incubation. In our assay, no obvious effect from bovine serum albumin on the activity of
the compounds has been found, which indicated that the new compounds selectively inhibited
the target enzyme but did not interact with bovine serum albumin.
Computational Modeling and CoMSIA Analysis: The crystal structure of AtHPPD was
taken from PDB data bank (PDB ID: 1TFZ). Compounds were constructed and optimized by
using SYBYL 7.0 (Tripos Inc.) and GasteigerꢀHuckel charges were calculated for them.
Docking calculations were performed on the two molecules by using AutoDock 4.0. The protein
and ligand structures were prepared with AutoDock Tools. A total of 256 runs were launched for
each molecule. Each docked structure was scored by the builtꢀin scoring function and was
clustered by 0.8 Å of RMSD criterions. The best binding modes were determined by docking
scores and also the comparison with available complex crystal structure of DAS645 with
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AtHPPD (PDB entry: 1TG5) as reference.28 Standard Amber ff99 force field parameters were
assigned to protein, and general AMBER force field (gaff) was assigned to ligands. The partial
atomic charges of ligands were calculated using the AM1ꢀBCC method and the system was
solvated in an octahedral box of TIP3P water with the crystallographic water molecules kept.
The edge of the box was at least 8 Å from the solute, appropriate counterions were added to the
system to preserve neutrality. In each step, energy minimization was first performed by using the
steepest descent algorithm for 1,000 steps and then the conjugated gradient algorithm for
another 2,000 steps.
We selected the majority of the new molecules which show different activities to perform
3DꢀQSAR. All molecular modeling and 3DꢀQSAR studies were performed using SYBYL7.3
with TRIPOS Force Field. The 3D structures of all compounds were built by using default
setting of SYBYL, and the molecules were subjected for energy minimization at a gradient of
1.0 kcal/mol with delta energy change of 0.05 cal/mol. The CoMFA descriptors, steric and
electrostatic field energies were calculated using the SYBYL default parameters: 2.0 Å rid
points spacing, an sp3 carbon probe atom with +1 charge and a minimum σ(column filting) of
2.0 kcal/mol, and the energy cutoff of 30.0 kcal/mol.
Herbicidal Activities. The postꢀemergent herbicidal activities of compounds I-VI, against
Echinochloa crusꢀgalli (EC), Setaria faberi (SF), Digitaria sanguinalis (DS), Amaranthus
retroflexus (AR), Eclipta prostrata (EP) and Abutilon juncea (AJ) were evaluated according to
the previously reported procedure;14ꢀ16, 29 the commercial triketone herbicide mesotrione was
selected as a positive control. All test compounds were formulated as 100 g/L emulsified
concentrates by using DMF as solvent and Tweenꢀ80 as emulsification reagent. The
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concentrates were diluted with water to the required concentration and applied to potꢀgrown
plants in a greenhouse. The soil used was a clay soil, pH 6.5, 1.6% organic matter, 37.3% clay
particles, and CEC 12.1 mol/kg. The rate of application (g ai/ha) was calculated by the total
amount of active ingredient in the formulation divided by the surface area of the pot. Plastic pots
with a diameter of 9 cm were filled with soil to a depth of 8 cm. Approximately 20 seeds of the
tested weeds were sown in the soil at the depth of 1ꢀ3 cm and grown at the temperature of 15ꢀ30
°C in a greenhouse. The air relative humidity was 50%. The diluted formulation solutions were
applied for postꢀemergence treatment. Broadleaf weeds were treated at the 2ꢀleaf stage and
monocotyledon weeds were treated at the 1ꢀleaf stage. The postꢀemergence application rate was
150 g ai/ha. Untreated seedlings were used as the control group and the solvent (DMF +
Tweenꢀ80)ꢀtreated seedlings were used as the solvent control group. Herbicidal activity was
evaluated visually at 15 days postꢀtreatment (Tables 1 and 2), with three replicates per treatment.
Crop Selectivity. The conventional rice, soybean, cotton, wheat, canola and maize were
planted separately in pots (12 cm diam.) containing test soil and grown in a greenhouse at 20ꢀ25
°C.23 After the plants had reached the 4ꢀleaf stage, the spraying treatment was conducted at the
dosage of 150 g ai/ha. The visual injury and growth state of the individual plants were observed
at regular intervals. After 15 days, the final results of crop safety were determined (Table 3),
using three replicates per treatment.
RESULTS AND DISCUSSION
Chemistry. According to the substituents at R2, the target compounds I-VI were prepared
by four different synthetic routes. When R2 is a hydrogen atom, the synthetic details for
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compound I are shown in Figure 1; when R2 are alkoxy or aryloxy groups, the synthetic routes
for compounds II and III are outlined in Figure 2; when R2 are methyl or trifluoromethyl groups,
the detail synthetic routes for compounds IV and V are shown in Figure 3; when R2 is a cyano
group, the detail synthetic routes for compounds VI are outlined in Figure 4. The lead
compound I can be obtained by a threeꢀstep synthetic route using the commercially available
quinolineꢀ3ꢀcarboxylic acid 1 as the starting material (Figure 1). The carboxylic acid 1 was very
sensitive to SOCl2, POCl3, PCl5, and PCl3; because of the high reactivity of the hydrogen at the
2 position on the quinoline ring. To our delight that, by using 1.2 equivalents of oxalyl chloride
as the chloroformylation reagent, CH2Cl2 as the reaction solvent, the corresponding acid
chloride can be successfully synthesized. The acid chloride thus obtained was very unstable, so
it was used in the next step without further purification. Subsequently, the acid chloride reacted
with 1,3ꢀcyclohexanedione, the enol ester 2 can be obtained in a yield of 78%. Finally, by using
acetone cyanohydrin as the Fries catalyst, the lead compound I was synthesized in a yield of
66%.
Since no carboxylic acids 7a-q are commercially available, the reported methods were
applied to synthesize them.30ꢀ32 As seen in Figure 2, the target compounds II and III were
synthesized by an eightꢀstep synthetic route using (substituted) anilines 3a-q as the starting
materials. After reaction with acetic anhydride, the corresponding (substituted)
Nꢀphenylacetamides 4a-q can be synthesized in high yields. Subsequently, the corresponding
intermediates 5a-q were obtained in yields of 30ꢀ80% via VilsmeierꢀHaack reaction. It was
found that, the substitutions of R3 have a big impact on reactivity at this reaction step, if R3 were
electronꢀwithdrawing groups, the reaction yields were very low or did not react at all. 5a-q were
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then oxidized by iodine in methanol, compounds 6a-q were synthesized in yields of 70ꢀ98%.
Various carboxylic acids 7a-q were prepared by a three step oneꢀpot method. More specifically,
6a-q first reacted with sodium alkoxides or aryloxides, and then water was added to the reaction
solution to hydrolyze the methoxycarbonyl group. After treating the residue with citric acid, the
corresponding 7a-q were obtained in yields of 64ꢀ98%. Followed by chloroformylation and
esterification reaction, the enol esters 8a-l and 9a-t were obtained in yields of 42ꢀ90%. Finally,
by using the same method as the synthesis of I, the title compounds II and III can be afforded in
yields of 52ꢀ84%.
The key intermediates 11a and 11b were prepared by reaction of methyl acetoacetate 10a or
ethyl trifluoroacetoacetate 10b with oꢀtoluidine 3j in the presence of 4ꢀmethylbenzenesulfonic
acid as the catalyst (Figure 3). Then, 11a and 11b reacted with N,NꢀDimethylformamide (DMF)
and POCl3 in dichloroethane (DCE), the corresponding 12a and 12b were obtained in yields of
90% and 50%, respectively. Alkaline hydrolysis of 12a and 12b with LiOH.H2O in the presence
of methanol and water furnished the hydroxyacids 13a and 13b. The synthesis of the target
compounds IV and V from 13a and 13b were the same as the preparation of compound I.
Similarly, compounds VI can be obtained by a sixꢀstep reaction route using methyl
2ꢀchloroꢀ8ꢀmethylquinolineꢀ3ꢀcarboxylate 6j as the starting material, after cyanation reaction
with CuCN, the key intermediate 16 was synthesized in a yield of 88%. Then followed by
hydrolysis, chloroformylation, enol esterification, and rearrangement reactions, the target
compounds VI can be successfully obtained.
The chemical structures of all of the synthetic triketoneꢀquinoline hybrids (I-VI) were
1
confirmed by H NMR, 13C NMR spectroscopic and HRMS spectrometric data. Furthermore,
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the structures of compounds III-d and III-f were verified by Xꢀray analysis (Figure 5).
HPPD Inhibition and SAR. The Ki value of the designed lead compound I against AtHPPD
was about 0.075 ꢀM, suitable worth for further optimization (Table 1). Molecular modeling was
performed to guide us in rational structural optimization. Because no crystal structure of
compound I with AtHPPD available, we used the reported binding mode of DAS645 with
AtHPPD (PDB entry: 1TG5) as reference.33 There were two major interactions of compound I
with AtHPPD active site (Figure 6). The triketone part of compound I can form a bidentate
interaction with the active site ironII, while the quinoline motif can form πꢀπ interaction with
Phe360 and Phe403. Furthermore, there was no significant interaction of compound I with
active site of AtHPPD (around 4Å residues); which indicated that a variety of substituent groups
might be introduced to compound I to increase its interaction with the enzyme. To obtain new
compounds with improved AtHPPD inhibitory activity we then designed compounds II-VI
based on the core structure on lead compound I.
The substituents at R1 have a significant impact on the activity (Table 1). In most cases,
compounds with a 5,5ꢀdiꢀCH3 at R1 had decreased HPPD inhibitory activity compared to lead
compound I. Although the Ki value of compound II-i was 0.041 ꢀM, it showed a slightly potent
activity than compound I, however, its activity is still far from enough when compared with
mesotrione. For further optimization, compound II-g was chosen as a representative to
investigate the binding modes. The trikeone part of compound II-g can form a bidentate
association with the ferrous iron active site and the quinoline ring can form πꢀπ interaction with
Phe360 and Phe403, respectively (panel A of Figure 7). When the two methyl groups were
introduced to the 5ꢀpositions of the 1,3ꢀcyclohexane ring, the distances between the methyl
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groups and the surrounding residues would be reduced to lead to steric hindrance. The distances
from two methyl groups on the cyclohexanedione ring of compound II-g to residues Asn261,
Ser246, Lys400, is 3.0 Å, 2.9 Å and 3.1 Å, respectively (left picture of Figure 7). The loss of
activity might due to the spatial repellence caused by the methyl groups at 5ꢀpositions of the
1,3ꢀcyclohexane ring. Therefore, we proposed that the removal of steric hindrance at this
position may be favorable to activity. After one methyl group was removed from this position,
the activity of compound II-k was greatly improved compared with its parent compound II-j,
however, its activity was still inadequate. This suggests that removing the remaining methyl
group would further improve the activity of these molecules.
Based on the above analysis, another methyl group was also removed from the 5ꢀposition of
the 1,3ꢀcyclohexane ring of compound II-j. The Ki value of compound III-a was as low as
0.009
ꢀM, about 10 times more potent than its parent compound II-k, slightly more potent than
commercial mesotrione (Ki = 0.013
ꢀM). Encouraged by these results, we further optimized the
structure and studied the activity of this series of compounds. As the steric effects at R1 had a
big impact on activity, compounds III-b—III-d were synthesized to test the contribution of R2
on activity. The sterically bulk groups at R2 were also detrimental to activity, for example,
compound III-a with a methoxy group at R2 displayed enhance activity relative to compounds
III-b—III-d (III-a > III-b > III-c > III-d). To understand the structural basis, compound III-d
was used as a representative for further binding modes investigation. The distance of benzyl
group between Phe371 is 3.0 Å, showing strong steric repulsion effect (Panel B of Figure 7).
On the basis of above observation, the methoxy group at R2 was retained and another set of
compounds III-eꢀIII-t were synthesized to further investigate their HPPD inhibitory activity
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(Figure 2). All newly synthesized compounds displayed inhibitory constants at the nanomolar
range, some of them even showing slightly more potential than mesotrione (Table 1). The
position of a single group at R3, has a big influence on activity. For example, when a methyl
group was introduced at 6ꢀposition (III-f), 7ꢀpositon (III-g) or 8ꢀpositon (III-a) on quinoline
ring, compound III-a would have better activity than compounds III-g and III-f (III-a > III-g >
III-f). The similar SAR can also be applied to those with methoxyꢀsubstituted analogues (III-i >
III-h) and chloroꢀsubstituted analogues (III-k > III-j). It appeared that the steric bulky groups
at R3 were also detrimental to activity (III-a > III-m > III-n). As to the multiꢀsubstituted
analogues, compounds with substituents at 7,8 positions showed improved activity relative to
those equivalent substitutions at 5,8 and 6,8 positions (III-q > III-p > III-o). It seemed that the
electronꢀwithdrawing groups at R3 would produce enhanced effects compared to those with
electronꢀdonating groups, although there may be few exceptions.
Herbicidal Activity and SAR. The postꢀemergence herbicidal activity of the newly prepared
compounds were tested against monocotyledon weeds (E. crusꢀgalli, S. faberii, and D.
sanguinalis) and broadleaf weeds (A. retroflexus, E. prostrata, and A. juncea) in the greenhouse
experiments. The commercial herbicide mesotrione was used as a control; the results are listed
in Table 1 and Table 2. The treated weeds had developed unique whitening symptoms in light,
which indicated that these compounds inhibited HPPD in planta. Some of the synthesized
compounds displayed promising and broadꢀspectrum herbicidal activities at the rate of 150 g
ai/ha. Compounds III-l even exhibited broader spectrum of weeds control (inhibition > 80%)
than mesotrione at the rate of 75 g ai/ha.
In most cases, introducing substitutions at R1 were detrimental to herbicidal activity (Table 1).
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Notably, compounds with sterically bulky groups at R2 also displayed decreased herbicidal
activity effects (III-a > III-6b > III-c > III-d). These results were consistent with in vitro
HPPD inhibitory activity. Furthermore, an interesting structureꢀactivity relationships was
observed with respect to the R3 substitution. For example, when a single substitution was
introduce at different positions on the quinoline ring, compounds with substituents at 7 position
would have broaderꢀspectrum of weeds control than those at 8ꢀ and 6ꢀ positions (III-g > III-a >
III-f). It seemed that bulky groups on the quinoline ring were favorable to herbicidal activity
(III-n > III-m > III-a). In most case, compounds with electronꢀwithdrawing groups at R3 were
found to exhibit higher herbicidal activity than those with electronꢀdonating groups (III-k >
III-g). Surprisingly, compounds II-d and III-l with methylthio group at R3 also displayed
excellent herbicidal activity at the rate of 150 ai/ha, even at a dosage as low as 37.5 g ai/ha
compound III-1 still showed over 80% control against tested broadleaf weeds and over 65%
inhibition against monocotyledon weeds (Table 2). A possible explanation for the enhanced
herbicidal activity of compounds II-d and III-l is that, the unique properties of methylthio group
make compounds more easily absorbed by plants.33
In this work, some of the synthesized compounds had good HPPDꢀinhibiting activity,
however, their herbicidal activities were not satisfactory. For example, compound III-a (Ki =
0.009 ꢀM) showed a slightly higher HPPD inhibitory activity than mesotrione (Ki = 0.013 ꢀM),
however, its herbicidal activity still could not compete with mesotrione. As far as we know,
there are many reasons for a compound having good in vitro inhibitory activity without
displaying promising herbicidal activity, such as, the absorption, distribution, metabolism, and
excretion (ADME) properties. As there is a methoxy group at the 2ꢀ position on quinoline ring
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of compound III-a. We inferred that methoxy group of compound III-a might be easily
metabolized by plants.34 This because the unique chemical properties of quinoline make its 2ꢀ
position very active compared with other positions. Therefore a series of compounds with
different metabolism blocking groups were synthesized to verify this hypothesis (Figure 3 and
4). As indicated in Table 1, among the newly prepared compounds IV and VI, compounds with
electronꢀwithdrawing group at R2 were found to detrimental to herbicidal activity. Promisingly,
compounds IV-a and IV-b displayed significantly enhanced herbicidal activity than their parent
compound III-a and II-k, respectively. Therefore, the SAR of R2 can be summarized as follows:
CH3 > CF3> OCH3 > CN > OCH2H3, OCH2CH2H3 >OCH2Ph.
Crop Selectivity. As mentioned in the introduction, crop selectivity is one of the main
concerns in herbicides discovery. Compounds III-l, III-m, and III-n with promising herbicidal
activity were chose as representatives for further crop selectivity studies. Maize displayed high
tolerance to compound III-l after postꢀemergence application at the rate of 150 g ai/ha (Table 3).
The promising results indicated that, compound III-l might be developed as a potential
herbicidal for maize fields. Most promisingly, compound III-m was found to be 'selective' to
maize, rice, and wheat at the dosage of 150 g ai/ha, however, the commercial mesotrione was
found to be 'nonseletive' to rice and wheat at the same rate. These results showed that there is a
great potential for III-m to be developed as a herbicide for weeds control in maize, rice, and
wheat fields. Furthermore, compound III-n was safe for maize at the dosage of 150 g ai/ha,
indicating that III-n might be developed as a potential herbicide for maize field.
CoMFA analysis. To further understand the substituent effects on AtHPPD inhibition, we
performed a brief CoMFA analysis of 31 representative compounds with SYBYL.29 The
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predicted CoMFA model was established with the conventional coefficient r2 = 0.974 and the
crossꢀvalidated coefficient q2 = 0.806, the predicted nonꢀcrossꢀvalidated coefficient r2(pred) =
0.854. The calculated activity values of the representative compounds are shown in Table 4, and
the plots of the predicted versus the actual activity values for the selected compounds are shown.
The contour map of the steric contributions was marked in green and yellow areas (Figure 8A).
The yellow contour means a bulky substituent in this area is unfavorable for the inhibition
activity of AtHPPD. For example, the compound II-b (R1 = 5,5ꢀdiCH3, Ki =0.304
ꢀM) is less
active than compound III-i (R1=H, Ki = 0.029 M), although they have the same R2 and R3
ꢀ
groups, which is similar as II-e and III-m, II-f and III-n, II-j and II-k. On the contrary, the
green region highlights positions where a bulky group would be favorable for HPPD inhibition
activity. The electrostatic contour map is shown in Figure 9B. The blue color represents that the
electroꢀpositive group will be advantageous to the bioactivity of HPPD inhibitors. The
electroꢀnegative groups is marked by red area, which are closed to the region of R2 group and
the 7,8ꢀpositions on the quinoline ring. So we can infer that the compound which bears an
electroꢀwithdrawing group at R2 and R3 (7 and 8 position) will show higher activity. The
experimental values are consistent with our calculation results, for example, the activity of III-r
(R3 = 7ꢀFꢀ8ꢀCH3, Ki = 0.011
7ꢀClꢀ8ꢀCH3, Ki = 0.014
M), III-b (R3 = OCH2H3, Ki = 0.025
0.171 M) and the activity of compounds VI-a and VI-b
M) > III-d (R3 = OCH2Ph, Ki = 0.213
ꢀ
M) > III-s (R3 = 7ꢀClꢀ8ꢀCH3, Ki = 0.014
ꢀ
M) > III-t (R3 =
ꢀ
ꢀ
M) > III-c (R3 = OꢀnꢀPr, Ki =
ꢀ
ꢀ
are higher than most of other compounds in Table 4, because of the cyano group at R2 position.
In conclusion, a series of novel triketoneꢀquinoline hybrids were rationally designed and
identified as potent HPPD inhibitors for herbicide discovery. Most of the synthesized
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compounds displayed excellent HPPD inhibitory activity, and some of them were even superior
to the commercial herbicide mesotrione. To our delight that, compounds III-l, III-m and III-n
displayed promising herbicidal activity at the rate of 37.5ꢀ150 g ai/ha. Especially, compound
III-l displayed broader spectrum of weed control than mesotrione. In addition, compounds III-l
and III-n were highly ‘selective’ to maize, III-m was safe for maize, rice, and wheat by
postꢀemergence application at the rate of 150 g ai/ha. These results indicated that
triketoneꢀquinoline hybrids could be novel lead compounds for novel herbicides discovery.
Further field trials and structure optimization of compounds III-l, III-m and III-n are
underway.
SUPPORTING INFORMATIONupporting Information
The detailed information for the preparation and the analytical data of compounds IꢀVI are
shown in the Supporting Information. These materials are available free of charge via internet at
http://pubs.acs.org.
398 ACKNOWLEDGMENTS
399
400
401
The authors are very grateful to the National Key Technologies R&D Program of China (No.
2011BAE06B03) and National Natural Science Foundation of China (No.21332004 and
21372093).
402
403
404
405
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Figure captions:
Figure 1. Synthetic route for lead compound I.
Reagents and conditions: (a) (COCl)2, CH2Cl2, ꢀ5 °C; (b) 1,3ꢀcyclohexanedione, CH2Cl2, 0 °C;
(c) acetone cyanohydrin, Et3N, CH2Cl2, RT
.
Figure 2. Synthetic route for compounds II and III.
Reagents and conditions: (a) (CH3C)2O, Et3N, CH2Cl2, 0 °CꢀRT. (b) DMF, POCl3, 0ꢀ80 °C; (c)
K2CO3, CH3OH, I2, reflux; (d) Na, alcohols, reflux; (e) H2O, citric acid; (f) (COCl)2, CH2Cl2, ꢀ5
°C; (g) (substituted) 1,3ꢀcyclohexanedione, CH2Cl2, 0 °C; (h) acetone cyanohydrin, Et3N,
CH2Cl2, RT
.
Figure 3. Synthetic route for target compounds IV-V.
Reagents and conditions: (a) pꢀtoluenesulfonic acid (pꢀTSA), cyclohexane, reflux; (b) DMF,
POCl3, DCE, 70ꢀ80 °C; (c) LiOH·H2O, methanol, H2O, reflux; (d) citric acid; (e) (COCl)2,
CH2Cl2, 0 °C; (g) (substituted) 1,3ꢀcyclohexanedione, Et3N, CH2Cl2, 0 °C; (h) acetone
cyanohydrin, Et3N, CH2Cl2, RT
.
Figure 4. Synthetic route for target compounds VI.
Reagents and conditions: (a) CuCN, DMF, reflux; (b) LiOH·H2O, methanol, H2O, reflux; (c)
citric acid; (d) (COCl)2, CH2Cl2, 0 °C; (e) (substituted) 1,3ꢀcyclohexanedione, Et3N, CH2Cl2, 0
°C; (f) acetone cyanohydrin, Et3N, CH2Cl2, RT
.
Figure 5. Xꢀray crystal structures for compounds III-d and III-f.
Figure 6. Simulated binding mode of lead compound I with AtHPPD and the designed
compounds II-III. The ferrous iron is shown in cyan sphere, the structure of compound I is
shown in yellow sticks, and the key residues surrounding the active site are shown in light blue.
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Figure 7. Simulated binding mode of compounds II-g (A) and III-t (B) with AtHPPD. The
ferrous iron is shown as a cyan sphere, the structures of compounds II-g and III-t are shown in
yellow sticks, and the key residues surrounding the active site are shown in light blue.
Figure 8. A: CoMFA map for steric contribution. Compound IV-b is shown inside the filed.
Green polyhedra represents the sterically favored areas where more bulky substituents favor
activity and yellow polyhedra represents the disfavored areas where less bulky substituents
favor activity. B: CoMFA map for electrostatic contribution. Compound IV-b is shown inside
the filed. Blue contours mean the increase of positive charge in these areas will promote the
activity; on the contrary, it will enhance the activity if we increase negative charges in the red
contours areas. Compound IV-b is shown inside the filed. C: The alignment of 24 compounds
of training set.
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Page 27 of 40
Journal of Agricultural and Food Chemistry
Table 1: Chemical Structures and PostꢀEmergent Herbicidal Activitya of Compounds I-IV, and
Their Inhibitory Activities Against AtHPPD.
AtHPPD
% Inhibition
Compd.
R1
R2
R3
Inhibition
ECb SFb DSb ARb EPb AJb
Ki (ꢀ
M)c
H
H
H
0
0
0
0
30
0
0
0
0
0
0
0
0
0
0
0
75
0
0
0
30
0
70
40
0
0
0
0
0
50
30
40
0
0
0
30
0
60
0
80
0
80
90
80
0
0
0
0
0
0
30
0
0.075±0.001
0.055±0.005
0.304±0.008
0.055±0.001
0.341±0.005
0.222±0.009
0.132±0.009
0.626±0.210
0.291±0.015
0.041±0.004
0.298±0.007
0.099±0.003
0.074±0.009
0.009±0.001
0.025±0.002
0.171±0.008
0.213±0.006
0.062±0.003
0.030±0.002
0.024±0.001
0.076±0.005
0.029±0.005
0.072±0.008
0.011±0.001
0.009±0.001
0.017±0.001
0.035±0.007
0.039±0.001
0.028±0.002
0.016±0.001
0.011±0.001
0.014±0.001
0.021±0.001
0.007±0.001
0.007±0.001
0.020±0.003
0.007±0.001
0.008±0.001
I
5,5ꢀdiCH3 OCH3
5,5ꢀdiCH3 OCH3
5,5ꢀdiCH3 OCH3
5,5ꢀdiCH3 OCH3
5,5ꢀdiCH3 OCH3
6ꢀOCH3
7ꢀOCH3
6ꢀCl
7ꢀSH3
8ꢀCH2CH3
II-a
II-b
II-c
II-d
II-e
II-f
II-g
II-h
II-i
II-j
II-k
II-l
III-a
III-b
III-c
III-d
III-e
III-f
III-g
III-h
III-i
III-j
III-k
III-l
III-m
III-n
III-o
III-p
III-q
III-r
III-s
III-t
IV-a
IV-b
V
80 80
40 15
45 30
70
0
5,5ꢀdiCH3 OCH3 8ꢀCH(CH3)2 50 40 45
5,5ꢀdiCH3 OCH3
5,5ꢀdiCH3 OCH3
5,5ꢀdiCH3 OCH3
5,5ꢀdiCH3 OCH3
5ꢀCH3
6,6ꢀdiCH3 OCH3
7ꢀFꢀ8ꢀCH3
7ꢀClꢀ8ꢀCH3
7ꢀBrꢀ8ꢀCH3
8ꢀCH3
0
0
0
0
0
0
0
0
0
0
15
0
0
0
30
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
50
OCH3
8ꢀCH3
8ꢀCH3
8ꢀCH3
8ꢀCH3
8ꢀCH3
8ꢀCH3
H
6ꢀCH3
50 50
30 50
20 80
30
30
0
20 100
0
60 80
0
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH2H3
OꢀnꢀPr
OCH2Ph
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
0
0
0
0
7ꢀCH3
6ꢀOCH3
7ꢀOCH3
6ꢀCl
7ꢀCl
7ꢀSCH3
8ꢀCH2CH3
0
50 30
95
0
55 95
90 100
90 100
90 85 90
75 80 60
OCH3 8ꢀCH(CH3)2 85 75 70
90 100 100
30
0
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
CH3
CH3
CF3
CN
CN
5,8ꢀdiCH3
6,8ꢀdiCH3
7,8ꢀdiCH3
7ꢀFꢀ8ꢀCH3
7ꢀClꢀ8ꢀCH3 70 60
7ꢀBrꢀ8ꢀCH3
8ꢀCH3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
H
H
H
H
40 50
80
90
70
60
90
60
0
80 90
80 80
30 70
90 80
90 95
50 80
30 30
0
0
75 60
80 85 80
50 30
0
0
5ꢀCH3
H
H
5ꢀCH3
8ꢀCH3
8ꢀCH3
8ꢀCH3
8ꢀCH3
0
0
0
0
0
VI-a
VI-b
0
0
0
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Journal of Agricultural and Food Chemistry
Page 28 of 40
mesotrione
85 50 75
95
95 95
0.013±0.001
b
aHerbicidal Activity was tested at the rate of 150 g ai/ha. Abbreviations: EC for Echinochloa
crusꢀgalli; SF for Setaria faberii; DS for Digitaria sanguinalis; AR for Amaranthus retroflexu;
c
EP for Eclipta prostrata and AJ for Abutilon juncea. Inhibition constant of the enzyme
reaction.
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Page 29 of 40
Journal of Agricultural and Food Chemistry
Table 2. PostꢀEmergent Herbicidal Activity of Compounds III-l to III-n, and IV-b.
% Inhibition
EC a SFa DS a AR a EP a AJ a
Compd.
dosage (g ai/ha)
75
37.5
75
37.5
75
37.5
75
37.5
75
80
70
70
60
80
70
80
60
80
50
80
65
65
45
60
45
75
50
30
30
80
70
40
30
60
30
70
30
70
55
80
80
80
80
80
80
85
75
80
80
80
80
80
80
90
80
80
75
90
90
90
85
90
80
85
80
80
70
90
85
III-l
III-m
III-n
IV-b
mesotrione
37.5
aAbbreviations: EC for Echinochloa crusꢀgalli; SF for Setaria faberii; DS for Digitaria
sanguinalis; AR for Amaranthus retroflexu; EP for Eclipta prostrata and AJ for Abutilon
juncea.
29
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